Using the system for genetic transformation and transgenic plant regeneration via somatic embryogenesis (SE) of Lycium barbarum established in this laboratory, this study reports the optimization of the factors affecting the efficiency of transformation, including pre-culture period, leaf explant source, use of acetosyringone, strains and density of Agrobacterium, and temperature of co-cultivation. The optimized transformation protocol for L. barbarum included pre-culture of leaf explants from 3-wk-old seedlings for 3 d on the medium for callus induction followed by inoculation with Agrobacterium strain EHA101 (pIG121Hm), co-cultivation for 3 d at 24°C, and transfer to the selection regeneration medium with 50 mg l⁻¹ kanamycin (Kan). Using this protocol, 65% L. barbarum explants gave rise to Kan-resistant and GUS-positive calli. In addition, the expression of introduced transgene (nptII) in clonal progeny was verified by formation of calli and somatic embryos from leaf segments of nine transgenic plants grown on the Kan-containing medium. All explants formed calli at 50 mg l⁻¹ Kan and seven out of nine transgenic plants were found to possess callus-forming capacity even at 100 mg l⁻¹ Kan. These calli also possessed higher SE potential on SE medium supplemented with 25 mg l⁻¹ Kan.